The calcium-sensing receptor (CaR) is a G protein-coupled, 7 transmembrane-spanning, cell surface receptor (GPCR) that binds calcium ions and allows cells to respond to changes in extracellular calcium concentrations. It plays a central role in systemic mineral metabolism by regulating parathyroid hormone secretion and renal calcium handling. In addition, it is expressed by many other cell types, including mammary epithelial cells (MECs). Expression of the CaR is low in rapidly proliferating MECs during pregnancy, but is greatly upregulated at the start of lactation. This allows the mammary gland to become a calcium-sensing organ that actively participates in the regulation of systemic calcium metabolism during lactation. In normal MECs, activation of the CaR inhibits PTHrP secretion but promotes calcium transport into milk. Therefore, if calcium delivery to the mammary gland falls, MECs transport less calcium and secrete more PTHrP. The increase in circulating PTHrP, in turn, activates bone resorption and liberates calcium from the skeleton for milk production. Thus, during lactation, a calcium-CaR-PTHrP mammary axis mimics the calcium-CaR-PTH parathyroid axis normally responsible for systemic calcium homeostasis. We have observed that malignant transformation alters the relationship between the CaR and PTHrP, such that breast cancer cells stimulate PTHrP production in response to calcium, rather than inhibiting PTHrP production, as they should. Similarly, activation of the CaR inhibits proliferation in normal cells, but stimulates proliferation in breast cancer cells. We hypothesize that these alterations in how the CaR regulates proliferation and PTHrP secretion in malignant versus normal breast cells contribute to the progression of breast tumors and the development of bone metastases. In order to investigate these possibilities, we outline three specific aims. Aim 1 will examine whether PKC phosphorylation of the CaR alters G-protein usage and cAMP and PTHrP production in response to calcium in malignant versus normal breast cells. Aim 2 will examine if disruption of the CaR gene will affect the development and progression of mammary tumors in mouse models. It will also determine if tumor CaR expression predicts outcome in a large cohort of patients with breast cancer. Aim 3 will examine if stimulation of PTHrP production by the CaR contributes to the development of osteolytic bone metastases in vivo.